The invention relates to animals in which the vitamin D receptor is misexpressed and methods of using such animals or cells derived therefrom, e.g., in methods of evaluating treatments for skin disorders, immune disorders and proliferation-related disorders.
The vitamin D receptor (VDR), is a nuclear receptor which heterodimerizes with the retinoid X receptor and interacts with specific DNA sequences on target genes, regulating their expression. The VDR is evolutionary well conserved and is expressed early in development in amphibians (Li Y. et al., (1997) Endocrinology 138:2347-2353), mammals (Johnson J. A. et al., (1996) J. Bone Miner. Res. 11:56-61), and birds (Elaroussi M. A. et al., (1994) Biochim. Biophys. Acta 1192:1-6; and Tuan R. S. and Suyama E., (1996) J. Nutr. 126:1308S-1316S). The VDR is expressed in tissues involved in mineral ion homeostasis, such as the intestine, the skeleton, and the parathyroid glands, as well as in tissues not thought to play a role in mineral ion homeostasis, such as the epidermis and the pancreas (Stumpf W. E. et al., (1979) Science 206:1188-1190).
Its principal ligand, 1,25-dihydroxyvitamin D is a steroid hormone that plays a role in mineral ion homeostasis. Insights into the physiological actions of 1,25-dihydroxyvitamin D have been obtained from studies in vitamin D deficient animals (Dostal L. A. and Toverud S. U., (1984) Am J. Physiol. 246:G528-G534; Halloran B. P. and DeLuca H. F., (1981) Arch. Biochem. Biophys. 209:7-14; Mathews C. H. E. et al., (1986) Am. J. Physiol. 250:E725-E730; Miller S. C. et al., (1983) Calcif. Tissue Int. 35:455-460; and Underwood J. L. and DeLuca H. F., (1984) Am. J. Physiol. 246:E493-E498) as well as in humans with VDR mutations (Balsan S. et al., (1986) J. Clin. Invest. 77:1661-1667; and Beer S. et al., (1981) Clin. Endocrinol. 14:395-402). These investigations have demonstrated that 1,25-dihydroxyvitamin D plays an important role in intestinal calcium absorption and that animals lacking in active hormone or its nuclear receptor develop hypocalcemia, rickets, osteomalacia, and hyperparathyroidism.
1,25-dihydroxyvitamin D is also believed to inhibit parathyroid hormone synthesis in the parathyroid gland (Silver J. et al., (1985) Proc. Natl. Acad. Sci. USA 82:4270-4273), blocking interleukin-2 production in activated lymphocytes (Manolagas S. C. et al., (1990) Kidney Int. 29:S9-S16), stimulating insulin secretion in the pancreas (Norman A. W. et al., (1980) Science 209:823-825), and decreasing proliferation and inducing differentiation of keratinocytes in the epidermis (Bikle D. D. et al., (1988) Endocrinology 124:655-660).
In general, the invention features, a non-human animal, in which the gene encoding the vitamin D receptor is misexpressed.
In preferred embodiments the animal, which is preferably a transgenic animal, is a mammal, e.g., a nonhuman primate or a swine, e.g., a miniature swine, a monkey, a goat, or a rodent, e.g., a rat, but preferably a mouse.
In preferred embodiments, expression of the gene encoding the vitamin D receptor is decreased as compared to the wild-type mouse. For example, the levels of the vitamin D receptor can be suppressed by, at least, 50, 60, 70, 80 or 90% or 100% as compared to the wild-type mouse.
In preferred embodiments, misexpression of the gene encoding the vitamin D receptor is caused by disruption of the vitamin D receptor gene. For example, the vitamin D receptor gene can be disrupted through removal of DNA encoding all or part of the receptor, e.g., removal of all or part of a zinc-finger domain, e.g., the second zinc-finger of the DNA-binding domain of the receptor.
In preferred embodiments, the animal can be heterozygous or homozygous for a misexpressed VDR gene, e.g., it can be a transgenic animal heterozygous or homozygous for a VDR transgene.
In preferred embodiments, the animal is a transgenic mouse with a transgenic disruption of the VDR, preferably an insertion or deletion, which inactivates the gene product.
In another aspect, the invention features, a nucleic acid sequence which, when introduced into an animal or cell, results in the misexpression of the VDR gene in the animal or cell. In preferred embodiments, the nucleic acid sequence, includes a VDR sequence which includes a disruption, e.g., an insertion or deletion and preferably the insertion of a marker sequence. For example, nucleic acid sequence can be the targeting construct, shown in FIG. 1.
In another aspect, the invention features, a method of evaluating a treatment for a skin disorder. The method includes: administering the treatment to a VDR misexpressing animal or a cell therefrom; and determining the effect of the treatment on a parameter related to the skin disorder, to thereby evaluate the treatment for the skin disorder. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal or cell.
In preferred embodiments, the skin disorder is a proliferative skin disorder. For example, the skin disorder can be a hyperproliferative skin disorder, e.g., psoriasis, squamus cell carcinoma, alopecia and the like.
In preferred embodiments, the effect of the treatment on the proliferative skin disorder can be determined by measuring such parameters as the thickness of the keratinocyte layer or the number and size of papules present on the skin of the animal; cell growth; tumor growth, weight or invasiveness; life span; tissue morphology; weight; or the expression of a gene related to cell proliferation.
In preferred embodiments, the effect of the treatment on alopecia can be determined by measuring such parameters as hair growth (in terms of the number, thickness, or growth rate of hairs), hair follicle morphology, or the pattern of hair growth.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment for an immune disorder or condition. The method includes: administering the treatment to a VDR misexpressing animal or a cell therefrom; and determining the effect of the treatment on a parameter related to the immune disorder or condition, to thereby evaluate the treatment for the immune disorder or condition. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal or cell.
In preferred embodiments, the effect of the treatment on the immune disorder or condition can be determined by measuring a parameter such as: the presence, function, or morphology of T cells or their progenitors; the presence, function, or morphology of B cells or their progenitors; the presence, function, or morphology of natural killer cells or their progenitors; resistance to infection; life span; body weight; the presence, function, or morphology of tissues or organs of the immune system; or the expression of a gene related to an immune disorder or condition.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment for promoting acceptance of a graft. The method includes: providing an animal, in which the gene encoding the vitamin D receptor is misexpressed; (optionally) introducing a graft tissue into the animal; administering the treatment to the animal; and determining the effect of the treatment on the acceptance of the graft by the animal to, thereby, evaluate the treatment for promoting acceptance of a graft.
The graft tissue can be an allograft or a xenograft. Allografts can be partially or fully mismatched. E.g., matched allografts can be matched at one or more class I locus, one or more class II locus, or one or more minor antigen locus. Xenografts can be primate, e.g., monkey, or swine, e.g., miniature swine, or dog grafts. Grafts, allografts or xenografts, can be an organ, e.g., a lung, kidney, heart, liver, or endocrine tissue, e.g., pancreatic tissue, e.g., pancreatic islets, connective tissue, bone, skin, or bone marrow.
In preferred embodiments, the animal is an animal described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal.
In preferred embodiments, the method further includes administering a second treatment to the animal. For example, an immunosuppressant, e.g., Azathioprine, Cyclosporine, Muromonab-CD3, FK506, rapamycin or Bromocriptine Mesylate can be administered with the treatment. The second treatment can be a treatment which induces tolerance to a graft, e.g., the introduction of donor bone marrow cells into the recipient of the graft.
In preferred embodiments, the effect of the treatment in promoting acceptance of a graft can be determined by measuring a parameter such as: acceptance of the graft; the presence, function, or morphology of T cells or their progenitors; the presence, function, or morphology of B cells or their progenitors; the presence, function, or morphology of natural killer cells or their progenitors; resistance to infection; life span; body weight; or the presence, function, or morphology of tissues or organs of the immune system; or the ability to exhibit immunological tolerance to the graft tissue.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment for an autoimmune disease. The method includes: administering the treatment to a VDR misexpressing animal or a cell therefrom; and determining the effect of the treatment on a parameter related to the autoimmune disease, to thereby evaluate the treatment for the autoimmune disease. The method may be performed in vivo or in vitro.
In preferred embodiments, the method further includes providing an animal or cell model for the autoimmune disease, e.g., a disorder described herein. A preferred animal model is the NOD mouse.
In preferred embodiments, the disorder is an autoimmune disease such as rheumatoid arthritis, e.g., juvenile rheumatoid arthritis, psoriatic arthritis, psoriasis, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, multiple sclerosis, allergic encephalomyelitis, systemic lupus erythematosus, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, scleroderma, Wegener""s granulomatosis, chronic active hepatitis, myasthenia gravis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn""s disease, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, diabetes, e.g., primary juvenile diabetes, dry eye associated with Sjxc3x6gren""s syndrome, uveitis posterior, or interstitial lung fibrosis.
In preferred embodiments, the animal or cell, is an animal or cell described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal or cell.
In preferred embodiments, the effect of the treatment on the autoimmune disease can be determined by measuring a parameter such as: the presence, function, or morphology of T cells or their progenitors; the presence, function, or morphology of B cells or their progenitors; the presence, function, or morphology of natural killer cells or their progenitors; resistance to infection; life span; body weight; or the presence, function, or morphology of tissues or organs of the immune system; acceptance of a graft, preferably a syngeneic graft, e.g., syngeneic pancreatic cells; or, generally, the ability of the animal or cell to recognize the animal""s or cell""s own antigens.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment for a condition characterized by unwanted cell proliferation, e.g., malignant growth. The method includes: providing an animal or a cell, in which the gene encoding the vitamin D receptor is misexpressed; (optionally) inducing or introducing a proliferative cell, e.g., a malignant cell, in the animal (or inducing a proliferative state, e.g., malignancy in a cell); administering the treatment to the animal or cell; and determining the effect of the treatment on a parameter related to a condition characterized by unwanted cell proliferation, to thereby evaluate a treatment for the condition characterized by unwanted cell proliferation. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal or cell.
In preferred embodiments, the effect of the treatment on the condition characterized by unwanted cell proliferation can be determined by measuring such parameters as the cell growth; tumor growth, weight or invasiveness; life span; tissue morphology; weight; or the expression of a gene related to unwanted cell proliferation.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment suitable for modulating, e.g., inhibiting or promoting, hair growth. The method includes: administering the treatment to a VDR misexpressing transgenic mouse, e.g., a knockout mouse; and determining the effect of the treatment on a parameter related to hair growth to, thereby, evaluate the treatment suitable for modulating hair growth. The evaluation can include determining the effect on: hair growth (in terms of the number, thickness, or growth rate of hairs), hair follicle morphology, or the pattern of hair growth.
In another aspect, the invention features, a method of evaluating a treatment for psoriasis. The method includes: administering the treatment to a VDR misexpressing transgenic mouse, e.g., a knockout mouse; and determining the effect of the treatment on a parameter related to psoriasis, to, thereby, evaluate the treatment for psoriasis.
In another aspect, the invention features, a method of treating hirsutism or hypertrichosis in a subject, e.g., a human. The method includes inhibiting the activity of VDR in the subject""s keratinocytes by, for example, disrupting the gene encoding the vitamin D receptor in the subject""s keratinocytes, or by administering, to the subject, a 1,25 dihydroxyvitamin D analogue, e.g., an antagonist, an anti-VDR antibody, e.g., an anti-VDR intrabody, or an anti-sense nucleic acid which inhibits the expression of the VDR.
In preferred embodiments, the gene encoding the vitamin D receptor is disrupted by introduction into the keratinocytes of the subject of a nucleic acid sequence, which when introduced into the subject""s keratinocytes, results in the misexpression of the VDR gene in the subject""s keratinocyres.
In another aspect, the invention features, a cell, or a purified preparation of cells, from a VDR misexpressing animal, e.g., a VDR misexpressing animal described herein. In preferred embodiments, the cell is a transgenic cell, in which the gene encoding the vitamin D receptor is misexpressed. The cell, preferably a transgenic cell can be a keratinocyte, a monocyte, a pancreatic cell or a lymphocyte.
In preferred embodiments, the cell is heterozygous or homozygous for the transgenic mutant gene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is an animal, e.g., a non-human mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a mouse, in which one or more, and preferably essentially all, of the cells of the animal include a transgene. The transgene is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, e.g., by microinjection, transfection or infection, e.g., by infection with a recombinant virus. The term genetic manipulation is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9crodentxe2x80x9d refers to all members of the phylogenetic order Rodentia.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, the term xe2x80x9cmisexpressionxe2x80x9d refers to a non-wild type pattern of gene expression. Expression as used herein includes transcriptional, post transcriptional, e.g., mRNA stability, translational, and post translational stages. Misexpression includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus. Misexpression includes any expression from a transgenic nucleic acid. Misexpression includes the lack or non-expression of a gene or transgene, e.g., that can be induced by a deletion of all or part of the gene or its control sequences.
As used herein, the term xe2x80x9cknockoutxe2x80x9d refers to an animal or cell therefrom, in which the insertion of a transgene disrupts an endogenous gene in the animal or cell therefrom. This disruption can essentially eliminate VDR in the animal or cell.
As used herein, the term xe2x80x9cmarker sequencexe2x80x9d refers to a nucleic acid sequence that (a) is used as part of a nucleic acid construct (e.g., the targeting construct) to disrupt the expression of the gene of interest (e.g., the vitamin D receptor gene) and (b) is used to identify those cells that have incorporated the targeting construct into their genome. For example, the marker sequence can be a sequence encoding a protein which confers a detectable trait on the cell, such as an antibiotic resistance gene, e.g., neomycin resistance gene, or an assayable enzyme not typically found in the cell, e.g., alkaline phosphatase, horseradish peroxidase, luciferase, beta-galactosidase and the like.
As used herein, xe2x80x9cdisruption of a genexe2x80x9d refers to a change in the gene sequence, e.g., a change in the coding region. Disruption includes: insertions, deletions, point mutations, and rearrangements, e.g., inversions. The disruption can occur in a region of the native vitamin D receptor DNA sequence (e.g., one or more exons) and/or the promoter region of the gene so as to decrease or prevent expression of the gene in a cell as compared to the wild-type or naturally occurring sequence of the gene. The xe2x80x9cdisruptionxe2x80x9d can be induced by classical random mutation or by site directed methods. Disruptions can be transgenically introduced. The deletion of an entire gene is a disruption. The disruption can, e.g., occur in a region of the VDR which mediates heterodimerization, in a region which mediated DNA binding, e.g., a zinc-finger domain, or in a region which mediates ligand binding. Preferred disruptions reduce VDR levels to about 50% of wild type, in heterozygotes or essentially eliminate VDR in homozygotes.
As used herein, xe2x80x9cadministering a treatment to an animal or cellxe2x80x9d is intended to refer to dispensing, delivering or applying a treatment to an animal or cell. In terms of the therapeutic agent, the term xe2x80x9cadministeringxe2x80x9d is intended to refer to dispensing, delivering or applying the therapeutic agent to an animal by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the intranasal or respiratory tract route. The term xe2x80x9cadministeringxe2x80x9d is further intended to refer to bringing the therapeutic agent into close proximity with a cell, such that the therapeutic agent can exert its effects on the cell.
As used herein, xe2x80x9cpurified preparationxe2x80x9d is a preparation which includes at least 10, more preferably 50, yet more preferably 90% by number or weight of the subject cells.
As used herein, the term xe2x80x9cpsoriasisxe2x80x9d is art recognized and refers to a chronic, occasionally acute, non contagious, relapsing skin disease, characterized by distinct reddish, slightly raised plaques or papules (small, round skin elevations) covered with scales. Psoriasis ordinarily involves the scalp, the elbows, the knees, the back, and the buttocks. Occasionally, the disease can be generalized. Psoriasis is the result of an abnormally high rate of mitosis in epidermal cells that may be related to a substance carried by the blood, a defect in the immune system or a virus.
As used herein, the term xe2x80x9calopeciaxe2x80x9d is art recognized and refers to the loss of hair, wool or feathers, e.g., baldness.
As used herein, the terms xe2x80x9chirsutismxe2x80x9d and xe2x80x9chypertrichosisxe2x80x9d are conditions characterized by excessive growth of hair. The conditions can occur in females and children, with a distribution of hair similar to that found in adult males. Hirsutism is the result of the conversion of vellus hair to large terminal hair as a result of higher than normal levels of androgens. Hirsutism may be familial or idiopathic. Hypertrichosis is a diffuse increase in body hair which may be idiopathic or may result from hormonal disorders or drugs.
The methods of the invention allow rapid and efficient screening of treatments for skin disorders, immune disorders and proliferation-related disorders.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The drawings are first briefly described.